This invention relates to arrays, particularly polynucleotide arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as xe2x80x9cfeaturesxe2x80x9d) are positioned at respective locations (xe2x80x9caddressesxe2x80x9d) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example all polynucleotide targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Biopolymer arrays can be fabricated by depositing previously obtained biopolymers (such as from synthesis or natural sources) onto a substrate, or by in situ synthesis methods. Methods of depositing obtained biopolymers include loading then touching a pin or capillary to a surface, such as described in U.S. Pat. No. 5,807,522 or deposition by firing from a pulse jet such as an inkjet head, such as described in PCT publications WO 95/25116 and WO 98/41531, and elsewhere. For in situ fabrication methods, multiple different reagent droplets are deposited at a given target location in order to form the final feature (hence a probe of the feature is synthesized on the array stubstrate). The in situ fabrication methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, and described in WO 98/41531 and the references cited therein for polynucleotides. The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides from nucleoside reagents on a support by means of known chemistry. This iterative sequence is as follows: (a) coupling a selected nucleoside through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (i.e. the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (xe2x80x9cdeprotectionxe2x80x9d) from the now substrate bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions such as ammonium hydroxide, in a known manner.
The foregoing chemistry of the synthesis of polynucleotides is described in detail, for example, in Caruthers, Science 230: 281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al., Nature 310: 105-110, 1984; and in xe2x80x9cSynthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivativesxe2x80x9d, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat. Nos. 4,458,066, 4,500,707, 5,153,319, 5,869,643, EP 0294196, and elsewhere. The phosphoramidite and phosphite triester approaches are most broadly used, but other approaches include the phosphodiester approach, the phosphotriester approach and the H-phosphonate approach. The substrates are typically functionalized to bond to the first deposited monomer. Suitable techniques for functionalizing substrates with such linking moieties are described, for example, in Southern, E. M., Maskos, U. and Elder, J. K., Genomics, 13, 1007-1017, 1992.
In the case of array fabrication, different monomers may be deposited at different addresses on the substrate during any one iteration so that the different features of the completed array will have different desired biopolymer sequences. One or more intermediate further steps may be required in each iteration, such as the conventional oxidation and washing steps in the case of in situ fabrication of polynucleotide arrays.
In array fabrication, the quantities of polynucleotide available are usually very small and expensive. Additionally, sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require use of arrays with large numbers of very small, closely spaced features. It is important in such arrays that features actually be present, that they are put down accurately in the desired target pattern, are of the correct size, and that the DNA is uniformly coated within the feature. If any of these conditions are not met within a reasonable tolerance, and the array user is not aware of deviations outside such tolerance, the results obtained from a given array may be unreliable and misleading. This of course can have serious consequences to diagnostic, screening, gene expression analysis or other purposes for which the array is being used. However, in any system used to fabricate arrays with the required small features, there is inevitably some degree of error, either fixed (and hence repeated) and/or random. The present invention realizes that in the special case of in situ fabrication method or any other method requiring deposition of multiple droplets at a target feature location, drop deposition errors from cycle to cycle may be different and are cumulative in determining errors in the finally formed features.
It would be desirable then to provide a means by which errors in features resulting during an in situ or any array fabrication method requiring multiple droplet deposition for a target feature location, can be readily determined.
The present invention further realizes that in array fabrication using the in situ method or any method using multiple drop deposition for a target feature location, the final error in a feature is a sum of errors during respective droplet depositions, and that such individual droplet errors may vary during each cycle at a given feature. Such errors may either be fixed or random. For example, in the fabrication of a polynucleotide array using phosphoramidite chemistry, different drop dispensers (such as pulse jets which eject drops drops toward a surface) may be provided to deposit different phosphoramidite droplets. Each nozzle may have its own inherent fixed drop dispensing error (such as error in drop dispenser position within a head, droplet size, or direction of drop). Also, the positioning system may have fixed inherent errors. Further, random errors can occur which are different during the dispensing of any one droplet. For example, air currents may vary during different droplet dispensing steps, or ambient temperature variations may cause expansion/contraction in dispensing apparatus components which affects absolute and/or relative positions of dispensed droplets. Thus, an error in the final feature may be a sum of errors from individual drop deposition at each feature. The present invention provides a means of tracking such individual droplet deposition error for a feature and using such multiple drop deposition error for that feature to determine an overall feature error.
In one aspect, the present invention provides a method which includes obtaining a set of multiple images of a target feature location on an array of multiple features. Each image of the set represents the target feature location following deposition of a corresponding sub-set of multiple droplets for that feature. An overlay composite is generated from the image set. Note that the processing for this can be done as each image of a set is obtained or after all images of a set are obtained. By an xe2x80x9coverlay compositexe2x80x9d in this context is referenced that information from at least one same location on the set of images is combined or compared using some function (for example, logical AND or some other operation). The same location may be, for example, all of the target feature location plus some predetermined area around that location, or just some point or portion of the foregoing. A particular overlay composite comprises a region of overlap of the multiple droplets. One or more feature characteristics (for example, the extent, size, shape or location of overlap of deposited droplets) may be provided by the overlay composite. Typically, since an array will usually contain many features and it will be desirable to determine feature characteristics of multiple such features (and perhaps all of them), the method may be used by obtaining multiple image sets of respective multiple target feature locations. In this case each image set represents a corresponding target feature location with each image of the set representing the location following deposition of a corresponding sub-set of multiple droplets for that feature. An overlay composite may then be generated from each of the image sets.
The above aspect may be used in the fabrication of arrays, or by an end user of the array or elsewhere. When the method is used in fabrication, it will also typically include depositing multiple reagent droplets for each of multiple target feature locations so as to form the array of features, and obtaining the image sets during such fabrication. In the fabrication, the image set is typically obtained by capturing an image of each target feature location following deposition of a corresponding drop sub-set (for example, from multiple captured images each of which simultaneously includes multiple target feature locations following a cycle of droplet depositions at those locations). When the above aspect is used by an end user or elsewhere, the image sets may be obtained by other means (such as from a portable data storage medium associated with the array and carrying the image sets as digital data, or from communication with a remote array fabricator).
The overlay composite can be used for any of a number of purposes. For example, the overlay composite may also be used by the fabricator as a quality control tool such as by rejecting arrays with one or more feature characteristics which do not meet predetermined requirements, and/or by altering the deposition of additional droplets for the feature, or of droplets for other features on the same or another array. Such rejecting or altering may be based at least in part on the overlay composite. Additionally, the overlay composite may be forwarded to a remote user of the array for use by them in interrogating the array or processing data resulting from the interrogation. Typically, the remote user will expose the array to a sample, interrogate the array following the exposure and optionally process the results of the evaluation (such as for evaluating the sample composition, for example, for the presence of a component). The interrogation or processing may be based at least in part on the overlay composite. However, in an alternative aspect of the present invention, rather than (or in addition to) forwarding the composite overlay to the remote user, the image sets themselves may be stored on a storage medium and forwarded to the remote user (such as by hard copy or on a portable digital data storage medium, or by communication). Any forwarding to the remote user may be, for example, be by hard copy or portable digital data storage medium carrying the required information, or by communication over a suitable communication channel.
The present invention also provides an apparatus for fabricating an array in accordance with any of the methods of the invention. The apparatus may include a drop deposition system to deposit multiple reagent droplets for each of multiple target feature locations so as to form the array of features. An image capture system provides the one or more sets of multiple images previously referenced. The apparatus may further include either a processor which generates an overlay composite from the image set and optionally performs any the processing previously described, or a storage medium onto which the image set or an overlay composite of them are stored, or both. Optionally, a communication module is present to communicate an image set or overlay composite to a remote location.
The present invention further provides a computer readable storage medium carrying program code which can be used with an apparatus of the present invention, to cause it to execute the steps of a method of the present invention.
The various aspects of the present invention can provide any one or more of the following and/or other useful benefits. For example, individual droplet deposition errors during an in situ or any array fabrication method requiring multiple droplet deposition for a target feature location, can be readily determined and tracked, and resultant feature errors determined from those individual droplet errors. Such results can be used as quality control tools during array fabrication and/or forwarded to a remote location for use in interrogating or processing interrogation results from the corresponding array.